Promoter Engineering Reveals the Importance of Heptameric Direct Repeats for DNA Binding by Streptomyces Antibiotic Regulatory Protein–Large ATP-Binding Regulator of the LuxR Family (SARP-LAL) Regulators in Streptomyces natalensis

[EN] The biosynthesis of small-size polyene macrolides is ultimately controlled by a couple of transcriptional regulators that act in a hierarchical way. A Streptomyces antibiotic regulatory protein–large ATP-binding regulator of the LuxR family (SARP-LAL) regulator binds the promoter of a PAS-LuxR...

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Detalles Bibliográficos
Autores: García Barreales, Eva, Morgado Vicente, Cláudia Sofia, Pedro López, Antonio de, Santos Aberturas, Javier, Aparicio Fernández, Jesús Manuel
Tipo de recurso: artículo
Estado:Versión publicada
Fecha de publicación:2018
País:España
Institución:Universidad de León
Repositorio:BULERIA. Repositorio Institucional de la Universidad de León
OAI Identifier:oai:buleria.unileon.es:10612/24068
Acceso en línea:https://journals.asm.org/doi/10.1128/aem.00246-18
https://hdl.handle.net/10612/24068
Access Level:acceso abierto
Palabra clave:Biología
Biotecnología
Antifungal agent
Gene regulation
LuxR
PAS domain
Polyene macrolide
SARP-LAL regulator
Streptomyces
2415.01 Biología Molecular de Microorganismos
2414.01 Antibióticos
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network_acronym_str ES
network_name_str España
repository_id_str
spelling Promoter Engineering Reveals the Importance of Heptameric Direct Repeats for DNA Binding by Streptomyces Antibiotic Regulatory Protein–Large ATP-Binding Regulator of the LuxR Family (SARP-LAL) Regulators in Streptomyces natalensisGarcía Barreales, EvaMorgado Vicente, Cláudia SofiaPedro López, Antonio deSantos Aberturas, JavierAparicio Fernández, Jesús ManuelBiologíaBiotecnologíaAntifungal agentGene regulationLuxRPAS domainPolyene macrolideSARP-LAL regulatorStreptomyces2415.01 Biología Molecular de Microorganismos2414.01 Antibióticos[EN] The biosynthesis of small-size polyene macrolides is ultimately controlled by a couple of transcriptional regulators that act in a hierarchical way. A Streptomyces antibiotic regulatory protein–large ATP-binding regulator of the LuxR family (SARP-LAL) regulator binds the promoter of a PAS-LuxR regulator-encoding gene and activates its transcription, and in turn, the gene product of the latter activates transcription from various promoters of the polyene gene cluster directly. The primary operator of PimR, the archetype of SARP-LAL regulators, contains three heptameric direct repeats separated by four-nucleotide spacers, but the regulator can also bind a secondary operator with only two direct repeats separated by a 3-nucleotide spacer, both located in the promoter region of its unique target gene, pimM. A similar arrangement of operators has been identified for PimR counterparts encoded by gene clusters for different antifungal secondary metabolites, including not only polyene macrolides but peptidyl nucleosides, phoslactomycins, or cycloheximide. Here, we used promoter engineering and quantitative transcriptional analyses to determine the contributions of the different heptameric repeats to transcriptional activation and final polyene production. Optimized promoters have thus been developed. Deletion studies and electrophoretic mobility assays were used for the definition of DNA-binding boxes formed by 22-nucleotide sequences comprising two conserved heptameric direct repeats separated by four-nucleotide less conserved spacers. The cooperative binding of PimRSARP appears to be the mechanism involved in the binding of regulator monomers to operators, and at least two protein monomers are required for efficient bindingSIThis work was supported by the Spanish Ministerio de Economía, Industria y Competitividad (grants BIO2013-42983-P and PCIN-2016-190 to J.F.A.), an F.P.U. contract of the Ministerio de Educación, Cultura y Deporte (FPU13/01537 to A.D.P.), and a contract from the Junta de Castilla y León cofinanced by the European Social Fund (to E.G.B.).American Society for MicrobiologyMicrobiologiaFacultad de Ciencias Biologicas y Ambientales2018info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttps://journals.asm.org/doi/10.1128/aem.00246-18https://hdl.handle.net/10612/24068reponame:BULERIA. Repositorio Institucional de la Universidad de Leóninstname:Universidad de LeónInglésinfo:eu-repo/grantAgreement/MINECO/Programa Estatal de Fomento de la Investigación Científica y Técnica de Excelencia/BIO2013-42983-Pinfo:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/PCIN-2016-190info:eu-repo/grantAgreement/MECD/Programa Estatal de Promoción del Talento y su Empleabilidad/FPU13http://creativecommons.org/licenses/by/4.0/info:eu-repo/semantics/openAccessoai:buleria.unileon.es:10612/240682026-06-24T12:43:27Z
dc.title.none.fl_str_mv Promoter Engineering Reveals the Importance of Heptameric Direct Repeats for DNA Binding by Streptomyces Antibiotic Regulatory Protein–Large ATP-Binding Regulator of the LuxR Family (SARP-LAL) Regulators in Streptomyces natalensis
title Promoter Engineering Reveals the Importance of Heptameric Direct Repeats for DNA Binding by Streptomyces Antibiotic Regulatory Protein–Large ATP-Binding Regulator of the LuxR Family (SARP-LAL) Regulators in Streptomyces natalensis
spellingShingle Promoter Engineering Reveals the Importance of Heptameric Direct Repeats for DNA Binding by Streptomyces Antibiotic Regulatory Protein–Large ATP-Binding Regulator of the LuxR Family (SARP-LAL) Regulators in Streptomyces natalensis
García Barreales, Eva
Biología
Biotecnología
Antifungal agent
Gene regulation
LuxR
PAS domain
Polyene macrolide
SARP-LAL regulator
Streptomyces
2415.01 Biología Molecular de Microorganismos
2414.01 Antibióticos
title_short Promoter Engineering Reveals the Importance of Heptameric Direct Repeats for DNA Binding by Streptomyces Antibiotic Regulatory Protein–Large ATP-Binding Regulator of the LuxR Family (SARP-LAL) Regulators in Streptomyces natalensis
title_full Promoter Engineering Reveals the Importance of Heptameric Direct Repeats for DNA Binding by Streptomyces Antibiotic Regulatory Protein–Large ATP-Binding Regulator of the LuxR Family (SARP-LAL) Regulators in Streptomyces natalensis
title_fullStr Promoter Engineering Reveals the Importance of Heptameric Direct Repeats for DNA Binding by Streptomyces Antibiotic Regulatory Protein–Large ATP-Binding Regulator of the LuxR Family (SARP-LAL) Regulators in Streptomyces natalensis
title_full_unstemmed Promoter Engineering Reveals the Importance of Heptameric Direct Repeats for DNA Binding by Streptomyces Antibiotic Regulatory Protein–Large ATP-Binding Regulator of the LuxR Family (SARP-LAL) Regulators in Streptomyces natalensis
title_sort Promoter Engineering Reveals the Importance of Heptameric Direct Repeats for DNA Binding by Streptomyces Antibiotic Regulatory Protein–Large ATP-Binding Regulator of the LuxR Family (SARP-LAL) Regulators in Streptomyces natalensis
dc.creator.none.fl_str_mv García Barreales, Eva
Morgado Vicente, Cláudia Sofia
Pedro López, Antonio de
Santos Aberturas, Javier
Aparicio Fernández, Jesús Manuel
author García Barreales, Eva
author_facet García Barreales, Eva
Morgado Vicente, Cláudia Sofia
Pedro López, Antonio de
Santos Aberturas, Javier
Aparicio Fernández, Jesús Manuel
author_role author
author2 Morgado Vicente, Cláudia Sofia
Pedro López, Antonio de
Santos Aberturas, Javier
Aparicio Fernández, Jesús Manuel
author2_role author
author
author
author
dc.contributor.none.fl_str_mv Microbiologia
Facultad de Ciencias Biologicas y Ambientales
dc.subject.none.fl_str_mv Biología
Biotecnología
Antifungal agent
Gene regulation
LuxR
PAS domain
Polyene macrolide
SARP-LAL regulator
Streptomyces
2415.01 Biología Molecular de Microorganismos
2414.01 Antibióticos
topic Biología
Biotecnología
Antifungal agent
Gene regulation
LuxR
PAS domain
Polyene macrolide
SARP-LAL regulator
Streptomyces
2415.01 Biología Molecular de Microorganismos
2414.01 Antibióticos
description [EN] The biosynthesis of small-size polyene macrolides is ultimately controlled by a couple of transcriptional regulators that act in a hierarchical way. A Streptomyces antibiotic regulatory protein–large ATP-binding regulator of the LuxR family (SARP-LAL) regulator binds the promoter of a PAS-LuxR regulator-encoding gene and activates its transcription, and in turn, the gene product of the latter activates transcription from various promoters of the polyene gene cluster directly. The primary operator of PimR, the archetype of SARP-LAL regulators, contains three heptameric direct repeats separated by four-nucleotide spacers, but the regulator can also bind a secondary operator with only two direct repeats separated by a 3-nucleotide spacer, both located in the promoter region of its unique target gene, pimM. A similar arrangement of operators has been identified for PimR counterparts encoded by gene clusters for different antifungal secondary metabolites, including not only polyene macrolides but peptidyl nucleosides, phoslactomycins, or cycloheximide. Here, we used promoter engineering and quantitative transcriptional analyses to determine the contributions of the different heptameric repeats to transcriptional activation and final polyene production. Optimized promoters have thus been developed. Deletion studies and electrophoretic mobility assays were used for the definition of DNA-binding boxes formed by 22-nucleotide sequences comprising two conserved heptameric direct repeats separated by four-nucleotide less conserved spacers. The cooperative binding of PimRSARP appears to be the mechanism involved in the binding of regulator monomers to operators, and at least two protein monomers are required for efficient binding
publishDate 2018
dc.date.none.fl_str_mv 2018
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv https://journals.asm.org/doi/10.1128/aem.00246-18
https://hdl.handle.net/10612/24068
url https://journals.asm.org/doi/10.1128/aem.00246-18
https://hdl.handle.net/10612/24068
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv info:eu-repo/grantAgreement/MINECO/Programa Estatal de Fomento de la Investigación Científica y Técnica de Excelencia/BIO2013-42983-P
info:eu-repo/grantAgreement/AEI/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/PCIN-2016-190
info:eu-repo/grantAgreement/MECD/Programa Estatal de Promoción del Talento y su Empleabilidad/FPU13
dc.rights.none.fl_str_mv http://creativecommons.org/licenses/by/4.0/
info:eu-repo/semantics/openAccess
rights_invalid_str_mv http://creativecommons.org/licenses/by/4.0/
eu_rights_str_mv openAccess
dc.publisher.none.fl_str_mv American Society for Microbiology
publisher.none.fl_str_mv American Society for Microbiology
dc.source.none.fl_str_mv reponame:BULERIA. Repositorio Institucional de la Universidad de León
instname:Universidad de León
instname_str Universidad de León
reponame_str BULERIA. Repositorio Institucional de la Universidad de León
collection BULERIA. Repositorio Institucional de la Universidad de León
repository.name.fl_str_mv
repository.mail.fl_str_mv
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score 15,81155

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